Abstract 5608: Deciphering role of NLRP3 inflammasome in acute myeloid leukemia microenvironment
Authors: Monalisa Sahoo, Manasi Nagare, Naythan Dcunha, Meena Patkar, Manju Sengar, Navin Khattry, Anant Gokarn, Sachin Punatar, Sridhar Epari, Tanuja Shet, Jyoti Kode
Journal: Cancer Research (2022)

Abstract Acute Myeloid Leukemia (AML), the most common form of leukemia in adults. Despite intensive chemotherapy regimen, approximately 60% of patients subsequently relapse. Inflammatory cells and mesenchymal stem cells (MSC) which are part of bone marrow (BM) microenvironment, play a critical role in cancer progression. Current strategies are focused on understanding cell-cell contact dependent/independent communication within the leukemic stem cell niche. NLRP3 is a part of innate immune mechanisms, resulting in the release of IL-1β and IL-18 immunomodulatory cytokines. Current study was aimed at analyzing NLRP3 pathway regulation at phenotypic and genotypic level in AML BM-MSC compared to healthy BM-MSC and their cross-talk with immune function in AML stem cell niche. MSC were isolated and cultured from AML BM that exhibited heterogenous fibroblastoid shape. Multi-color flow cytometry of AML BM-MSC revealed characteristic high expression of CD90 and minimal expression of CD45. They also expressed immune checkpoint markers TIM3, PDL-1, adenosine receptors A1R/A2BR, vimentin and NLRP3. Cultured AML BM MSC and fresh leukemic blasts demonstrated tunneling nanotubules, increased vesicles and increase in mitochondria which were of irregular shapes and sizes as seen by transmission electron microscopy. Microarray data revealed genes of NLRP3 damage-associated molecular patterns (DAMP) pathway viz. Nlrp3, NME8, Pycard and IL-18 were distinctly upregulated in AML BM-MSC compared to uninvolved BM-MSC. AML peripheral blood (PB) plasma showed immunosuppressive effect on healthy peripheral blood mononuclear cells. NK cell cytotoxicity was found to be significantly reduced in presence of culture supernatant of AML cell line and further reduced with supernatant of cells activated for NLRP3 pathway. Immunofluorescence demonstrated NLRP3 expression in AML BM-MSC and also in vesicles released from MSC. NLRP3 pathway activation was observed in AML cell line which was significantly abrogated upon treatment with NLRP3 inhibitor. NLRP3 pathway is stimulated using basic DAMP agent LPS and secondary activators ATP or Nigericin. Activation was visualized by colocalization of NLRP3 and caspase-1, in the presence/absence of NLRP3 inhibitor. Expression of IL-1β was observed using immunofluorescence and its release in culture supernatants was measured by ELISA. Inhibition of NLRP3 corroborated with decreased secretion of IL-1β in AML cell line. Thus, a positive correlation of IL-1β release was observed with expression of NLRP3. Paraffin sections of AML BM are being evaluated for NLRP3 pathway markers by immunohistochemistry. Understanding remodelling of BM-MSC, leukemia target and immune cell dynamics through NLRP3 pathway regulation in stroma microenvironment may provide crucial leads in therapeutic modulation of the immune response within the stem cell niche. Citation Format: Monalisa Sahoo, Manasi Nagare, Naythan Dcunha, Meena Patkar, Manju Sengar, Navin Khattry, Anant Gokarn, Sachin Punatar, Sridhar Epari, Tanuja Shet, Jyoti Kode. Deciphering role of NLRP3 inflammasome in acute myeloid leukemia microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5608.